Fabrication of an Effective Avermectin Nanoemulsion using a

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Agricultural and Environmental Chemistry

Fabrication of an Effective Avermectin Nanoemulsion using a Cleavable Succinic Ester Emulsifier Wenxun Guan, Liming Tang, Yan Wang, and Haixin Cui J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01388 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

Fabrication of an Effective Avermectin Nanoemulsion using a Cleavable Succinic Ester Emulsifier Wenxun Guan a, Liming Tang a,*, Yan Wang b, Haixin Cui b

[a]

Key Laboratory of Advanced Materials of Ministry of Education of China Department of Chemical Engineering Tsinghua University, Beijing, China (100084) E-mail: [email protected]

[b]

Institute of Environment and Sustainable Development in Agriculture Chinese Academic of Agriculture Sciences, Beijing 100081, China

1

ACS Paragon Plus Environment


1

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Abstract:

2

In this study, a new emulsifier precursor was prepared via esterification of avermectin with

3

succinic anhydride. The chemical structure of the product was confirmed to be mono-substituted

4

avermectin. After neutralization with triethanolamine, it exhibited adequate emulsification ability

5

for avermectin. Avermectin was then encapsulated in nanoparticles in the nanoemulsion with a

6

high drug loading up to 60 wt%, and high stability. The nanoemulsion of nanoparticles which

7

serves as a carrier of avermectin, showing high-efficient pesticide characteristics, including low

8

surface tension, high affinity to leaves, and improved photostability. In the presence of esterase or

9

under strongly basic condition, the ester bonds of the emulsifier can be hydrolyzed and the

10

encapsulated avermectin molecules can be released in an accelerated manner. Besides, the

11

nanoemulsion exhibited improved insecticidal effect compared with commercial emulsifiable

12

concentrate (EC), which was attributed to the cleavage of ester bonds of the emulsifier by esterase

13

in vivo.

14

Key word: avermectin • nanotechnology • nanopesticide • drug delivery • ester bond

2


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1

Introduction

2

Challenged by the rapidly growing global population and limited resources, researchers

3

are looking for highly efficient and environmentally friendly advanced materials and novel

4

ways of manufacturing. In agriculture, the application of pesticides has become one of the

5

most important approaches in controlling crop pests and diseases, to improve agricultural

6

production efficiency to fulfill the burgeoning food demand.

7

pesticide contributes up to 50% of crop gain in developing countries. [2] Though we have

8

become adept to the traditional pesticides that have been cultivated as highly-efficient and

9

economic, sometimes we forgot the efficiency of pesticides is still extremely limited, less

10

than 0.1% in most cases [3], and the majority of pesticides leaches into the surrounding

11

environment, triggering serious pollutions of water, soil and atmosphere.

12

due to commercial pesticides are usually applied in the form of emulsifiable concentrate

13

(EC), suspending agents or wettable powder [6,7] along with a large amount of organic solvent

14

or other additives, leading to a variety of problems, such as coarse particles, poor water

15

dispersibility, low bioactivity, and slow degradation.

[1]

It has been reported that

[4,5]

This is mainly

[8]

16

Spurred by the successful application of nanotechnology in the field of medicine

17

development of novel pesticides has now been closely coupled with nanotechnology to

18

improve performances in recent decades [9,10]. Nanopesticide systems, relying on extremely

19

high surface area nanomaterials to adsorb or encapsulate pesticide, are designed to enhance

20

dispersibility and stability of pesticides in water, adhesion to crops and bioactivity, as well as

21

to enable controlled releasing.

22

developed [12-13] in the form of nano-microemulsion, nanocapsules and nanosuspensions by

[11]

,

During the past decade, various nanopesticides have been

3



[14]

[15,16]

25

the present methods are non-trivial, expensive and usually involve with extra

26

non-biodegradable components.

[23]

silica,

[20]

aluminum,

copper oxide,

silver,

[19]

24

[22]

titanium dioxide,

[18]

using lipids,

zinc oxide,

emulsifier,

[17]

23

[21]

polymer,

Page 4 of 33

and carbon nanotubes [24]. However, most of

27

Controlled releasing, ubiquitously seen in drug delivery systems, is a unique technique

28

that can deliver molecules to the target position with minimal loss through reliable and

29

rapidly-transporting “carriers”, which later release the “cargo” upon external stimuli. The

30

controlled manner of this technique greatly benefits the transportation of drugs with much

31

enhanced efficacy, prolonged effective time and reduced side effects. Controlled releasing in

32

drug delivery systems has been studied for decades and a variety of well-developed

33

approaches have been reported

34

such as temperature,

[25]

magnetic fields,

35

[30]

[31]

or enzymes.

36

temperature-triggered unfolding of a leucine zipper peptide to conduct a transporting

37

channel in the membrane of a doxorubicin (Dox)-carrying liposome for the releasing of

38

doxorubicin. Xiao, Z. and coworkers

39

dehybridization of DNA conjugates to delivery doxorubicin from the surface of gold

40

nanorods.

redox reaction,

[8]

through numerous endogenous and exogenous stimuli, [26]

ultrasounds,

[27]

light,

[32]

[28]

electric fields,

. Al-Ahmady, Z. S. and coworkers

[34]

[33]

[29]

pH,

used a

used near-infrared-triggered induction of

41

In analogue to the application in drug delivery systems, controlled releasing has also

42

been widely-used in pesticides to prolong duration, reduce application amount, and suppress

43

side effects. [35] Sanghamitra Atta and his coworkers

44

conjugate, and evaluated photolytic release of the pesticide 2, 4-D under visible light. Yubin

[36]

synthesized perylene 2, 4-D ester

4


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45

Xiang and his coworkers [37] utilized the nanocarrier micro-nano pores of diatomite/Fe3O4 to

46

load pesticide, within which the nanopesticide was coated by chitosan, a key ingredient for

47

controlled releasing of pesticide in acid condition. Disregarding the rich library of

48

controlling releasing methods for nanopesticides,

49

suffering from high cost and complexity during preparation.

[36-39]

current technologies are still

50

In order to obtain a more practical and effective nanopesticide system, we show our

51

efforts to design a common reversible covalent bond, the ester bond, as a suitable cleavable

52

moiety in controlled releasing system in this work, due to its simplicity in synthesis.

53

design a simple and efficient emulsifier from all biodegradable molecules for avermectin.

54

Avermectin is one of the most important pesticides in agriculture for animal husbandry and

55

human parasitic diseases, but the low water solubility (~ 7.8 µg/mL) and poor photostability

56

extensively limit its application.

57

successfully embedded the hydrophobic avermectin into hydrophilic succinic acid, and the

58

resultant emulsifier has adequate emulsification ability for avermectin, which can help form

59

stable avermectin nanoemulsion after being neutrualized. In the presence of esterase or

60

under strongly basic condition, the emulsifier instantly releases the drug upon the cleavage

61

of the ester bond.

62

characteristics comparing with past literature reports (cite literatures here), including simple

63

synthesis, high drug loading (up to 60%), high stability, strong affinity to plant leaves, high

64

insecticide efficiency and good controlled releasing performances. Therefore, our method

65

could be further extended to other types of pesticides, and would inspire more effective

66

designs in the development of emulsifiers for nanopesticides.

[41,42]

[40]

We

Through a facile esterification reaction, we

[43]

. Our nanoemulsion system exhibits a series of advantageous

5



Page 6 of 33

67 68

Experimental section

69

Materials and instruments

70

Avermectin (MYM biological technology company, 95%), succinic anhydride

71

(Shanghai Macklin Biochemical Co. Ltd., 98%), 4-dimethylaminopyridine (Shanghai

72

Macklin Biochemical Co. Ltd., 99%), pyridine (Shanghai Macklin Biochemical Co. Ltd.,

73

AR), triethanolamine (Shanghai Macklin Biochemical Co. Ltd., AR), Esterase Pseudomonas

74

fluorescens, recombinant from E. coli (Sigma-Aldrich), phosphotungstic acid (Shanghai

75

Macklin Biochemical Co. Ltd, AR), avermectin EC (Hebei Weiyuan Chemical Co. Ltd., 5

76

wt%).

77

Microscopic melting point tester (X-4, Beijing TECH Instrument CO. LTD),

78

Fourier-transform IR spectrometer (FT-IR) (Nicolet 560), nuclear magnetic resonance

79

spectrometer (JNM-ECA 600), elemental analyzer (Vario EL Ⅲ ), Zetasizer (3000HS

80

Malvern), transmission electron microscopy (TEM) (Hitachi H600), surface tension meter

81

(HZ800, Zibo Bo Hai Apparatus Mountain Arsenal), UV-vis spectrophotometer (UV-3200,

82

Mapada instruments).

83

Synthesis and characterization of avermectin derivative

84

The synthetic procedure of avermectin derivative as the emulsifier precursor was

85

detailed below. First, avermectin (1.774 g, 2 mmol) and excessive succinic anhydride (0.603

86

g, 6 mmol) were dissolved in pyridine (10 mL) in a three-necked flask, and

87

4-dimethylaminopyridine (DMAP, 0.072 g, 0.6 mmol) was added as a catalyst. The reaction

88

mixture was then heated at 50 ± 2 °C for 6 h under mechanical stirring. After cooling to 6


Page 7 of 33


89

room temperature, a pale yellowish solution of avermectin derivative was obtained. Then the

90

avermectin derivative solution was dropped on a silica gel column and was separated using

91

gradient elution method by ethyl acetate: petroleum ether = 1:7~1:1. The resulting solution

92

was rotary evaporated to remove solvent. After drying under vacuum, avermectin derivative

93

was obtained as a yellow solid in a yield of about 66%. Microscopic melting point tester was

94

used to test the melting range of the avermectin derivative from beginning to melting to

95

complete melting. FT-IR spectrometer, nuclear magnetic resonance spectrometer,

96

matrix-assisted

97

(MALDI-TOF-MS) and elemental analyzer were applied to characterize the chemical

98

structure of avermectin derivative.

99

Fabricating of avermectin nanoemulsion

laser

desorption

ionization

time

of

flight

mass

spectrometry

100

First, 0.12 g of triethanolamine (0.8 mmol) was added to 1.200 g of avermectin

101

derivative solution (with 0.210 g of avermectin derivative containing 0.4 mmol carboxylic

102

acid groups in pyridine solution) in a glass bottle under stirring for neutralization. Then,

103

0.250g of avermectin was dissolved in the solution. The solution was added slowly to

104

deionized water (18 mL) under moderate stirring (600 rpm) at room temperature. The

105

resulting nanoemulsion was dialyzed in water for 12 h to remove any water-soluble small

106

molecules and organic solvents. Then the nanoemulsion was transferred to a beaker. After

107

the addition of deionized water to a total weight of 25 g, the avermectin nanoemulsion was

108

obtained as pale yellow.

109

Characterization of the nanoemulsion

110

The size of the particles in the nanoemulsion was evaluated by dynamic light scattering 7



111

(DLS, 3000HS Malvern Zetasizer). A four-sided cuvette was filled with deionized water.

112

Drops of the nanoemulsion were added to the cuvette until the liquid looked blue. Then, the

113

cuvette was placed in the instrument and measured.

114

The morphology of the particles was imaged by using TEM. A few drops of the

115

nanoemulsion, which was diluted with deionized water 50 times, were deposited on a

116

carbon-coated copper grid. The excess solution was removed with filter paper. The samples

117

were then stained with 2% phosphotungstic acid solution (the pH value was adjusted to 7~8)

118

for 60 s. The excess dye was removed with filter paper, and the copper grids were dried at

119

room temperature. The sample was imaged using Hitachi H600 TEM operated at 80 keV.

120

The particle size and size distribution were obtained based on the particle sizes of 168

121

particles counted from ten randomly selected regions in ten different TEM photographs.

122

Stability of the nanoemulsion

123

The stability of avermectin nanoemulsion was evaluated by four methods. The first is

124

dilution stability. The avermectin nanoemulsion was diluted 100 times or 1500 times using

125

deionized water, and the particle sizes and PDI were determined by DLS. The second is

126

centrifugal stability. The nanoemulsion was divided and transferred to identical centrifuge

127

tubes. After centrifugation under 10 000 rpm (7826 g) for 5, 10, 15, 20, 25, and 30 min, the

128

particle sizes and PDI in the supernatant were measured by DLS. The third is temperature

129

stability, the nanoemulsion was divided and sealed in clean brown vials in the refrigerator

130

(0 °C), the room temperature desiccator (25 °C) and 54 °C incubator, respectively. After 2, 4,

131

6, 8, 10, 12 and 14 days, the particle sizes and PDI were determined by DLS. The final is

132

photostability. Different concentration of the ethanol solutions of pure avermectin and

Page 8 of 33

8


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133

avermectin encapsulated with emulsifier were prepared separately. The absorption curves of

134

the two samples were measured by an UV−vis spectrophotometer. A certain amount of

135

avermectin ethanol solution and avermectin nanoemulsion was dripped and spread in the

136

petri dishes separately. Then these petri dishes were dried in the vacuum oven, placed under

137

an UV lamp (1000 W), and irradiated for a pre-designed time period. After that, the sample

138

was dissolved in ethanol and analysed by an UV−vis spectrophotometer to determine the

139

concentration of unresolved avermectin.

140

Retention and contact angle on leaves

141

The amount of liquid retention on the surface of the plant leaves was measured by the [44]

142

micro weighing method and the dipping method.

Specifically, the liquid sample and

143

elongated tip tweezers were placed in the beaker and weighed with a balance. Then, the

144

balance was zeroed. Leaves were cut into pieces, and areas S (cm2) of the pieces were

145

measured. One leaf piece was immersed in the liquid with tweezers for 15 s, then quickly

146

pulled out, and hung over the liquid, until no liquid drops were dripping. Then, the leaves

147

were placed aside, the tweezers were put back into the beaker, and the balance reading W (g)

148

was recorded. Leaf retention is calculated by 1000 × W/S (mg/cm2).

149

The contact angles of the avermectin nanoemulsion on cucumber and cabbage leaves

150

were measured by the precision contact angle measuring instrument. The mass concentration

151

of the active ingredient in the liquid was set as 0.02% according to the current applied

152

concentration in the field. Specific operations are as follows: fresh plant leaves were

153

collected and fixed on clean glass slides, avoiding damage to the foliage structures and

154

keeping the foliage in their natural state. Place the slides on the operation stage of contact 9



Page 10 of 33

155

angle measuring instrument. A drop of avermectin nanoemulsion was dripped onto the flat

156

surface of leaves through the micro syringe. The droplets on the leaves were photographed

157

using the CCD camera on the contact angle measuring instrument at 30s after dripping. The

158

contact angles of droplets on the experimental foliage were calculated by five-point fitting

159

analysis. The measurement temperature was 25 ± 2 °C, and the relatively humidity was 35%

160

± 2%. Each sample was tested five times at different locations on the foliage to reduce the

161

error caused by the surface differences of the leaves.

162

Surface tension measurement and fluorescence measurement

163 164

The critical micelle concentration (CMC) of the emulsifier was measured in two methods: surface tension method and fluorescence method.

165

The first is the surface tension method. Different amounts of avermectin derivative and

166

an excess of triethanolamine (2 times the molar amount of carboxyl groups in avermectin

167

derivative) were dissolved in the deionized water to prepare an aqueous solution of

168

emulsifier with different concentrations (5~1×10-5 g/L). The surface tension of the solution

169

was measured by the surface tension meter. Then the critical micelle concentration can be

170

determined by the curve of surface tension.

171

Then is the fluorescence method. A certain amount of pyrene solution in acetone were

172

added to the test tubes, and the acetone was evaporated naturally. The aqueous solution of

173

emulsifier with different concentrations (5~1×10-5 g/L) was added to the tubes containing

174

pyrene. These tubes were placed in a shaker for at least four hours to ensure the pyrene can

175

enter the hydrophobic micro domains. Then Fluorescence spectroscopy was used to

176

determine the luminescent properties of the emulsifier solution. Then the critical micelle 10


Page 11 of 33


177

concentration can be determined by calculating the value of I1/I3, which is the ratio of the

178

fluorescence intensity of the first electron vibration peak 373 nm (I1) to the third electron

179

vibration peak 384 nm (I3), at different concentrations.

180

Measurement of the releasing performance The drug releasing profile of the nanoemulsion was measured by the dialysis method.

181 182

[45]

183

and placed into a wide-mouth bottle. The ethanol and water (1:7) mixture (the pH was

184

adjusted to 8 with triethanolamine) was added to the bottle. The bottle was kept at room

185

temperature (25 °C) with magnetic stirring at 100 rpm. At a predesigned time interval, a 200

186

µL solution was withdrawn from the bottle and diluted with the releasing medium to 2 mL.

187

The diluted sample was analyzed by an UV−vis spectrophotometer to determine the

188

concentration of avermectin. The releasing profile of nanoemulsion at other conditions (pH

189

12 and pH 8 with 0.3 mg/ml esterase) and control groups (pure avermectin and avermectin

190

EC) were also measured by similar experiments.

191

Toxicological experiments of the pesticides

A total of 10 mL of nanoemulsion was transferred to a dialysis bag. The bag was sealed

192

Toxicological experiments were carried out according to the FAO recommended aphid

193

spray method. The specific experimental procedure was as followed: the concentration of the

194

drug (ppm, mg / L) is set to 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0, and the

195

corresponding deionized water without chemicals is blank control, and every treatment was

196

repeated for three times. The fresh cabbage leaves which had no contact with any drug were

197

washed and dried, and labelled with a hole puncher with a diameter of 6 cm wafer. Put two

198

layers of filter paper in a Petri dish and add a small amount of deionized water to moisten it. 11



Page 12 of 33

199

Then put the front of the blade on a petri dish with a diameter of 6 cm. Wingless healthy

200

aphids were carefully picked and put on the leaves with brush, 30 per dish. Then these

201

dished were sprayed accurately under the potter spray tower (spray pressure of 100kPa, each

202

treatment spray volume of 3 mL, settling time of 30 s), sealed with plastic membrane, and

203

the membrane was punctured a number of holes with a breathable pinhole. The treated

204

aphids were placed in a thermostatic incubator under the conditions of T (temperature) = (25

205

± 2) °C, L/D = 16: 8, RH = (75 ± 5) %. After 48 h, the mortality was calculated by counting

206

the dead and live insects using dissection microscope. Live insects were sentenced if their

207

feet or antennae quiver. If it is difficult to determine the survival of aphids, insect needle was

208

used. Those without any response under touch with insect needle were sentenced to death.

209

Then toxicity regression equations, correlation coefficients, LC50 (lethal concentration 50%,

210

the dose required to kill half the members of a tested population after a specified test

211

duration) and their 95% confidence intervals were calculated using DPS v12.01 statistical

212

software.

213

Results and discussion

214

Synthesis and characterization of the avermectin derivative

215

From practical point of view, the emulsifiers for nanopesticides should be cheap, easily

216

prepared and possess specific functions. In this article, an avermectin derivative was

217

prepared simply via the reaction of avermectin and succinic anhydride with the synthetic

218

reaction illustrated in Scheme 1. Although there are three hydroxyl groups in avermectin

219

molecule, the tertiary hydroxyl at C7 is inactive because of steric hindrance, and only the

220

secondary hydroxyl groups at C4″and C5 are capable of reacting with succinic anhydride. 12


Page 13 of 33


221

[46]

222

(their molar ratio was kept at 1:3) in pyridine at 50 ± 2 °C using 4-dimethylaminopyridine as

223

a catalyst. After reaction, the crude sample was purified by column chromatography to give

224

pure avermectin derivative with a yield of about 66%. Melting point apparatus, FT-IR

225

spectrometer, 1H-NMR spectrometer and elemental analyzer were applied to characterize the

226

chemical structure of the product.

To minimizing side reaction, avermectin was reacted with excessive succinic anhydride

227

The melting point of the avermctin derivative was determined to be 103~105 °C, which

228

was significantly lower than that of pure avermectin (150~155 °C). The melting range was

229

quite narrow.

230

The FT-IR spectra of pure avermectin and avermectin derivative were recorded and are

231

compared in Figure 1. In the spectrum of avermectin, the characteristic peaks of hydroxyl

232

groups appear at 3415~3550 cm−1 and 1618~1637 cm−1. However, these two peaks are

233

significantly lower in the spectrum of avermectin derivate, indicating that some hydroxyl

234

groups have been reacted. Instead, three broad and scattered absorption peaks appeared in

235

the range of 2500~2800 cm−1, representing the stretching vibration of O-H in carboxyl

236

groups. Instead of a single peak at 1730 cm−1 for C=O of the ester groups of avermectin, two

237

clear peaks appeared at 1740 cm−1 and 1720 cm−1 for avermectin derivate, representing the

238

carbon-oxygen double bonds of carboxyl groups and ester groups, respectively.

239

The 1H-NMR spectra of pure avermectin and avermectin derivative are shown in Figure

240

S1. The peak at 12.1 ppm could be attributed to hydrogen atoms of carboxylic acid groups in

241

avermectin derivative. The peak at 5.2 ppm, which is attributed to the hydrogen in hydroxyl

242

groups, is observed in both samples. However, this peak of pure avemectin is obviously 13



Page 14 of 33

243

larger than that of avermectin derivative. The peaks at 4.02 ppm and 3.13 ppm represent the

244

hydrogen atoms at C5 and C4’’ (the carbon atom which hydroxyl group is attached) of pure

245

avemectin. They partially transferred to 4.41 ppm and 3.55 ppm in avermectin derivative

246

after the formation of the ester bond. The peak at 2.37 ppm, representing the hydrogen atoms

247

on the methylene carbon in succinate segment, remains in avermectin derivative.

248

The elemental analysis is shown in Table 1. It is clear that the content of each element

249

in the product is consistent with the calculated value obtained from mono-substituted

250

avermctin. Since the activity of secondary hydroxyl groups in avermectin is low, and the

251

reaction conditions were relatively mild, mono-substituted avermectin was obtained as the

252

main product considering the yield (~66%). We assumed that both hydroxyl groups at C4″

253

and C5 of avermectin participated in the reaction because of their quite similar activities.[47,9]

254

Besides mono-substituted avermectin, di-substituted avermectin should also be formed

255

in the reaction. The MALDI-TOF-MS analytic results (Figure S2) confirmed that both

256

products were formed with mono-substituted avermectin as the main product. The ratio of

257

peak intensities of mono-substituted avermectin and di-substituted avermectin is about 2.3:1.

258

Fabrication and characterization of the avermectin nanoemulsion

259

After being neutralized by triethanolamine, the avermectin derivative could be used as

260

the emulsifier for avermectin because of its amphiphilicity and drug affinity. When the

261

solution, containing avermectin and the neutralized avermectin derivative that served as the

262

emulsifier, was dropped into deionized water under stirring, avermectin nanoemulsion was

263

formed spontaneously under electrostatic repulsion. After that, the resulting sample was

264

dialyzed in water to remove the remaining small molecules, giving a pure nanoemulsion. 14


Page 15 of 33


265

Without avermectin, the emulsifier itself could dissolve in the water completely.

266

The influences of triethanolamine and drug loading on the particle sizes of the resulting

267

nanoemulsions are summarized in Table 2. When the molar ratio of emulsifier and

268

triethanolamine was at 1:2, the particle size was rather large (198 nm) due to an insufficient

269

ionization of carboxylic groups. However, the particle sizes changed to 72.4 nm, 69.2 nm

270

and 70.1 nm at molar ratios of 1:3, 1:4 and 1:5, respectively, while the PDI (polydispersity

271

index) decreased gradually as increasing the molar ratios of emulsifier and triethanolamine.

272

Moreover, the particle sizes became larger and the PDI increased gradually as increasing the

273

drug loadings because more and more hydrophobic avermectin molecules were encapsulated.

274

The resultant nanoemulsions could keep stable for more than three months even at the drug

275

loading of 60%. Unless otherwise indicated, the nanoemulsion sample 3 was used in the rest

276

of investigation.

277

The morphology of the nanoparticles in the nanoemulsion was observed by

278

Transmission Electron Microscopy (TEM). From the results in Figure 2(a) and 2(b), the

279

particles were all with a spherical shape. On the basis of TEM images, the particle size and

280

size distribution were obtained. As shown in Figure 2(c), most particles are in the range of

281

50−80 nm, indicating relative uniform size distribution. The average particle size is

282

calculated to be 66.8 nm, smaller than the Z-average particle size of 72.4 nm.

283

Stability of the avermectin nanoemulsion

284

To meet the criteria of practical applications where nanoemulsions are usually in diluted [47]

285

solution prior to their use as pesticide,

we further tested the stability of nanoemulsion

286

against dilution. According to the using standard of commercially available avermectin EC 15



Page 16 of 33

287

(Beijing Green Agricultural Science and Technology Group CO. Ltd. Active ingredient

288

content: 1.8%, diluting 2000-fold when using), the dilution experiments of our

289

nanoemulsion (Active ingredient content 1.3%) with 100-fold and 1500-fold deionized

290

water were carried out. As shown in Figure S3, the photograph revealed significant exterior

291

change of the nanoemulsion upon dilution, including improved transparency and a

292

decreasing of particle size from 74.5 ± 2.5 nm to 65.4 ± 1.0 nm when diluted 100-fold and

293

63.2 ± 1.8 nm when diluted 1500-fold. So our nanoemulsion stayed nearly intact during

294

water dilution with minimal change of its physicochemical properties, and we believe the

295

slight size change of the particles would have neglect effect on its pesticide performances as

296

illustrated in the following discussion.

297

The centrifugal stability of the nanoemulsion was then assessed in a centrifugal

298

accelerated sedimentation experiment. We found the appearance and transparency of the

299

nanoemulsion merely changed after centrifugation at different time intervals. The particle

300

sizes remained in the range of 75-85 nm and the PDI remained around 0.25 as indicated in

301

Figure 3a, suggesting the avermectin nanoemulsion are mechanically stable against strong

302

shearing treatment.

303

The thermal stability of the nanoemulsion was evaluated by storage at different

304

temperatures for different time periods. Figure 3b shows the evolution of particle sizes as a

305

function of storage time. We find that after storage at lower temperature (4 °C or 25 °C) for a

306

long period, e.g. 15 days, the particle sizes only slightly increased from around 70 nm to

307

around 80 nm, while the PDI stayed nearly constant at about 0.170. However, as a sharp

308

contrast, the particles become much more unstable at a higher temperature of storage at 16


Page 17 of 33


309

54 °C for 15 days, as indicated by the significant dilation of particle size from 80 nm to

310

around 200 nm (Figure 3b). This proved that the avermectin nanoemulsion was quite stable

311

at 4 °C or 25 °C, which could be attributed to the strong charge repulsion caused by the large

312

surface area in the nanoemulsion. However, the nanoparticles dilated from 80 nm to around

313

200 nm after storage at 54 °C for 15 days, and the nanoemlusion turned to opaque. This

314

instability may be originated from prone to aggregation of the particles in the emulsion

315

under higher temperature, due to the accelerating movement of the particles and dissolution

316

of emulsifier molecules into water. [48]

317

Avermectin is a photosensitive pesticide.

[49]

It is necessary to investigate the

318

photostability of avermectin in its form of encapsulation in the nanoparticles. As clearly

319

suggested from the results in Figure S4, pure avermectin degraded faster than that in the

320

nanoemulsion. The emulsifier molecules located at the outer layer of the nanoparticles could

321

absorb UV light before the interior avermectin molecules because of their similar UV

322

absorption spectra as shown in Figure S5 due to their similar structures. Therefore, the

323

photostability of avermectin could be improved in the nanoemulsion.

324

Surface tension measurement and fluorescence measurement

325

A low surface tension is a desired property for spreading and wetting of nanoemulsion

326

droplets over the plant leaves, thus preventing the droplets from slipping. The results of the

327

surface tension test of the aqueous solution of the emulsifier at different concentrations are

328

shown in Figure 4a, which exhibits a typical surface tension profile of surfactants.

329

surface tension of pure deionized water was measured to be 71.0 mN/m at 25 °C. In the

330

lower emulsifier concentration range, the surface tension of the liquid decreases significantly

[50]

The

17



Page 18 of 33

331

with the increase of the emulsifier concentration. After passing through a critical

332

concentration, the surface tension plateaus at a value of 37.6±1.3 mN/m. According to this

333

measurement, the critical micelle concentration (CMC) of the emulsifier is determined to be

334

about 0.38 g/L.

335

We also applied the fluorescence measurement to further determine the CMC of the

336

emulsifier. Fluorescence emission spectra of different concentrations of the aqueous

337

solutions of the emulsifier containing pyrene as a fluorescent probe were measured. The

338

ratio of I1/I3 is calculated and plotted in Figure 4b as a function of concentration. As

339

indicated in the figure, pyrene molecules clearly underwent a transition from a polar water

340

environment to non-polar hydrophobic environment at the emulsifier concentration of 0.4

341

g/L, suggesting the formation of emulsifier micelles. This concentration could be regarded

342

as the CMC of emulsifier, and was close to the value determined by the surface tension

343

measurement.

344

Retention and contact angle on leaves

345

During the spraying process, the pesticide is firstly deposited on the crop leaf and then

346

transport to the rest of the plant for performing its pesticide function. A high affinity of the

347

pesticide liquid to the leaves is desired for reducing the loss and enhancing the efficacy. A

348

few studies have been reported to assist avermectin nanoparticles adhere to the leaf surface

349

by using polydopamine or polyurethane, but the preparation process of polydopamine or

350

polyurethane was non-trivial. [51,52]

351

In this study, the retention and contact angle on leaves were measured to understand the

352

affinity of pesticide liquid on the leaves. The results in Figure 5 shows that the retention on 18


Page 19 of 33


353

highly hydrophobic rice leaves (surface tension = 29.9 mN/m) of the nanoemulsion is 1.7

354

and 7.4 times the commercial avermectin EC and deionized water, respectively, and on

355

moderate hydrophobic cabbage leaves (surface tension = 38.9 mN/m) is 1.6 and 2.2 times

356

the commercial avermectin EC and deionized water, respectively, while on hydrophilic

357

cucumber leaves (surface tension = 58.7 mN/m) is 1.5 and 1.6 times the commercial

358

avermectin EC and deionized water, respectively. These results demonstrate that the

359

nanoemulsion has strong affinity on both hydrophilic and hydrophobic leaves, which is

360

likely owing to the lower surface tension of the emulsifier. Moreover, the improvement of

361

the adhesion ability is more significant on the hydrophobic leaves.

362

Several types of leaves have low surface tension, due to the thick wax layers, tomenta,

363

or mastold microstructure on the surface. It is a challenge to well-spread the nanoemulsion

364

droplet under such circumstances. With the emulsifier, the surface tension of the

365

nanoemulsion could be lowered to 37.6 mN/m. The contact angles on the cabbage and

366

cucumber leaves of the nanoemulsion droplet and water were measured and summarized in

367

the Figure 6. As seen from the figure, the contact angles on cabbage and cucumber leaves of

368

the avermectin nanoemulsion are 57.7° and 46.3°, respectively, smaller than those of water

369

(105.9° and 78.8°), showing the satisfying spreading ability of the nanoemulsion.

370

Drug releasing profile

371

The releasing performances of nanopesticides are another essential factor in their

372

application. As we discuss earlier, controlled releasing of pesticide molecules is preferable.

373

Among many ways of controlled releasing, reversible covalent bonds, such as disulfide bond,

374

imine bond and ester bond, are often used due to their reversible fracture characteristics. [42] 19



[51]

Page 20 of 33

375

In our previous work,

an amphiphilic disulfide-containing polyurethane was used as an

376

emulsifier to prepare avermectin nanopesticide with high photostability, efficacy, and much

377

improved controlled releasing behavior comparing to past reports. In this work, we use the

378

ester bonds as cleavable moieties in the emulsifier to promote the controlled releasing

379

profile.

380

To investigate the releasing behavior of the nanoemulsion, the nanoemulsion was

381

incubated in the mixture of water and ethanol (7:1) at pH 8 with or without esterase, or at pH

382

12. For comparison, we also measured the releasing profiles of the control samples

383

(including pure avermectin and avermectin EC) at the condition of pH 8 and 25°C. As

384

shown in Figure 7, the releasing rates of nanoemulsion at all conditions were slower than

385

that of pure avermectin, demonstrating that nanoemulsions have a certain sustained releasing

386

effect. The avermectin was released to the environment quite slowly for both avermectin EC

387

and the nanoemulsion at pH 8 without esterase, indicating the great chemical tolerance of

388

the samples. Moreover, the nanoemulsion could release avermectin in an accelerated rate

389

with the help of esterase or at pH 12.

390

It has been suggested that the dynamic ester bond could be hydrolyzed in the presence [53-54]

391

of esterase or under strongly basic condition.

As the breakage of ester bond under

392

specific condition, the emulsifier would lose its amphiphilicity. As a result, the particles

393

gradually became unstable in water due to the lack of surface charge stabilization. They

394

aggregated together into large-sized particles until precipitates appeared in the system as

395

shown in Figure S6. In such case, avermectin could be released in an accelerated rate. Since

396

esterase also exists in insect body, the controlled release of avermectin from nanoemulsion 20


Page 21 of 33


397

would become feasible when ester bond was designed into the emulsifier molecule. [55-56]

398

Toxicological properties

399

The drug releasing performance of nanopesticide has great influence on the insecticide

400

effect. The insecticide effect could be improved if the pesticide could be released in the

401

insect body.

402

The Myzus persicae Sulzer experiments were applied to determine the virulence of

403

avermectin nanoemulsion, commercial formulation EC, and the emulsifier itself. The

404

insecticidal effect of a pesticide is typically evaluated by the LC50 value.

405

Table 3, the LC50 value of the present nanoemulsion was only 20.25 µg/mL, significantly

406

lower than that of EC (38.20 µg/mL). The relative virulence of nanoemulsion and

407

avermectin EC (LC50 of avermectin EC/ LC50 of avermectin nanoemulsion) was calculated

408

to be 1.8, which is obviously higher than those of some reported avermectin nano pesticides

409

systems. [16,51,58]. In addition, the LC50 value of the neat emulsifier was determined to be

410

398.11 µg/mL, suggesting that the emulsifier has a rather low insecticidal effect. The high

411

insecticidal ability of the current nanoemulsion could be attributed to the breakage of ester

412

bonds in emulsifier molecules by active enzyme like esterase in vivo. As the emulsifier lost

413

its amphiphilicity, the nanoemulsion became unstable and the particles aggregated together

414

into large-sized precipitates. As a result, the active ingredient could be released in an

415

accelerated manner and play the role.

[57]

As shown in

416

In summary, the newly prepared avermectin derivative was demonstrated as an

417

emulsifier to form avermectin aqueous nanoemulsion after being neutralized. The resulting

418

nanoemulsion exhibited a series of exquisite performances. In the presence of esterase or 21



Page 22 of 33

419

under strongly basic condition, the ester bonds in the emulsifier could be hydrolysed,

420

therefore promoting the releasing of avermectin. The nanoemulsion also exhibited improved

421

insecticidal effect compared with commercial EC. Considering the simple fabrication

422

procedure, multifunctional designing in emulsifiers, well controlled releasing fashion and

423

improved insecticidal ability, this investigation would be helpful for designing more

424

effective emulsifiers of nanopesticides.

425

Acknowledgements

426

This work was financially supported by the National Basic Research Program of China

427

(973 Plan, 2014CB932202), the National Natural Science Foundation of China (21574074),

428

and the Fund of the Key Laboratory of Advanced Materials of Ministry of Education

429

(2017AML08).

22


Page 23 of 33


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57 Van F Kees. Insecticidal activity of Bacillus thuringiensis crystal proteins, J. Invertebr. Pathol., 2009, 101(1),1-16. 58 Zhang H, Qin H, Li L X, Zhou X T, Wang W, Kan C Y. Preparation and characterization of controlled-release avermectin/castor

oiled-based

polyurrthane

nanoemulsions.

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27



Page 28 of 33

Figures and tables

Scheme 1. Synthetic procedure for avermectin derivative

Figure 1. FT-IR spectra of the avermectin derivative and avermectin 28


Page 29 of 33


Figure 2. TEM images (a, b) of the particles and particles size distribution (c, particle sizes of 168 particles were counted from ten randomly selected regions in ten different TEM photographs) of the nanoemulsion.

Figure 3. Change in particle sizes and PDI of the nanoemulsion after centrifugal treatment (a, 3 samples at 10 000 rpm (7826 g)) or storage at different temperature. (b, 3 samples at 4°C, 25°C and 54°C, respectively)

29



Page 30 of 33

Figure 4. (a). Surface tension at different concentration of the emulsifier aqueous solution. (b) The value of I1/I3 of pyrene at different concentration of the emulsifier aqueous solution.

Figure 5. Leaf retention of avermectin nanoemulsion, avermectin EC and deionized water. (Retention was measured by counting the residual amount of liquid on each kind of leaves with 1 cm×1 cm surface area. Data is averaged over five samples for each type of leaf, respectively.)

30


Page 31 of 33


Figure 6. Contact angles of avermectin nanoemulsion and deionized water on cabbage and cucumber leaves

Figure 7. Avermectin releasing profiles for different avermectin formulations at different conditions. (at 25°C and pH 8 if not specially marked)

31



Page 32 of 33

Table 1. Elemental analysis of avermectin derivative Elemental Content (%) Measured values Calculated values based on mono-substituted avermectin

C

H

O

64.5±0.3

7.8±0.1

27.6±0.1

64.2

7.8

27.9

Table 2. Formulation of the emulsifier and particle size of the nanoemulsion Ratio of emulsifier and triethanolamine

Drug loading

Z-average particle size

(%)

of the dispersion (nm)

1

1:2

50%

198.9±7.9

0.361

2

1:3

50%

69.2±3.5

0.391

3

1:4

50%

72.4±5.5

0.263

4

1:5

50%

70.1±1.8

0.227

5

1:4

33%

49.2±1.6

0.180

6

1:4

60%

133.3±5.3

0.389

Sample No.

PDI

Table 3. Toxicity test results of different pesticides Pesticides

Toxicity regression a

LC50

95% confidence b

intervalc

equation

(µg/mL)

Avermectin nanoemulsion

Y=3.4497+1.1866x

20.26

15.83-25.55

Avermectin EC

Y=3.2734+1.0914x

38.20

29.20-51.90

Emulsifier itself

Y=2.824+0.8372x

398.11

196.62-616.75

a. Toxicity regression equation represents the relationship between log doses and lethality values. b. LC50 represents lethal concentration 50%, the dose required to kill half the members of a tested population after a specified test duration. c. 95% confidence interval of LC50.

32


Page 33 of 33


Table of content

33


Fabrication of an Effective Avermectin Nanoemulsion using a

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